Use of cinnabarinic acid as a modulator of immune response in autoimmune disorders

ABSTRACT

The present invention provides compounds and methods for preventing and treating an immune disorder in a subject.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplication No. 61/307,358, filed on Feb. 23, 2010, which isincorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant Nos. DP1OD000329 and R37 AI40312 awarded by the National Institutes of Health.The Government has certain rights in the invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

NOT APPLICABLE

BACKGROUND OF THE INVENTION

Ligation of Aryl Hydrocarbon Receptor (AHR) by environmental toxins hasbeen linked to numerous clinically relevant settings ranging fromautoimmune disorders to cancers. Therefore, there is a need to identifyand/or to develop compounds that are capable of treating these disordersrelated to Aryl Hydrocarbon Receptor (AHR). The present inventionsatisfies this and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compounds and methods for preventing andtreating an immune disorder in a subject.

In some embodiments, the methods comprise administering to the subjectcompounds of the present invention in an amount sufficient prevent ortreat an autoimmune disorder such as multiple sclerosis, myastheniagravis, Guillan-Barre syndrome (antiphospholipid syndrome), systemiclupus erytromatosis, Behcet's syndrome, Sjogrens syndrome, rheumatoidarthritis, Hashimoto's disease/hypothyroiditis, primary biliarycirrhosis, mixed connective tissue disease, chronic active hepatitis,Graves' disease/hyperthyroiditis, scleroderma, chronic idiopathicthrombocytopenic purpura, diabetic neuropathy and septic shock.

In one embodiment, the present invention provides a method of treatingan autoimmune disorder, the method comprising administering to a subjectin need thereof, a therapeutically effective amount of a compound offormula Ib:

wherein each R¹ is independently selected from the group consisting ofhydrogen, —NH₂, C₁₋₆ alkylamine, —CO₂H, C₁₋₆ alkyl-CO₂H, and C₀₋₆alkyl-C(O)NH₂ and each R² is independently selected from the groupconsisting of hydrogen, —NH₂, C₁₋₆ alkylamine, —CO₂H, C₁₋₆ alkyl-CO₂H,and C₀₋₆ alkyl-C(O)NH₂. Subscript m of formula 1b is an integer from 1to 3, and subscript n is an integer from 1 to 4. The compounds offormula 1b include salts and isomers thereof.

In another embodiment, the present invention provides a method ofmodulating Aryl Hydrocarbon Receptor activity by administering acompound of formula Ib:

wherein each R¹ is independently selected from the group consisting ofhydrogen, —NH₂, C₁₋₆ alkylamine, —CO₂H, C₁₋₆ alkyl-CO₂H, and C₀₋₆alkyl-C(O)NH₂ and each R² is independently selected from the groupconsisting of hydrogen, —NH₂, C₁₋₆ alkylamine, —CO₂H, C₁₋₆ alkyl-CO₂H,and C₀₋₆ alkyl-C(O)NH₂. Subscript m of formula 1b is an integer from 1to 3, and subscript n is an integer from 1 to 4. The compounds offormula 1b include salts and isomers thereof.

In other embodiments, the methods comprise administering CinnabarinicAcid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that Cinnabarinic Acid inhibits the development ofexperimental allergic encephalomyelitis (EAE) in C57BL/6 mice with asingle dose given at the time that MOG peptide and CFA were injected—Day0.

FIG. 2 illustrates that Cinnabarinic Acid activates AHR responsive(Dioxin Responsive Elements) in an in vitro test demonstrating agonistactivity. Tryptamine is a positive control and a downstream relatedmolecule (Compound 1) is a negative control.

FIG. 3 illustrates that Cinnabarinic Acid displaces Dioxin (TCDD) in anin vitro assay using purified in vitro translated human AHR proteinthereby demonstrating direct binding.

FIG. 4 illustrates that Cinnabarinic Acid potently induces IL-22 invitro in cultured human naïve CD4+ T cells stimulated withanti-CD3/anti-CD28 under “Th17” polarizing conditions (IL-1b, IL-21,IL-23 with anti-IFNg and anti-IL4). This is AHR dependent as inclusionof an AHR antagonist fully abrogated effect.

FIG. 5 illustrates that Cinnabarinic Acid inhibits the induction of EAEas compared to DMSO.

FIG. 6: 3-HKyn and 3-HAA promote IL-22 expression by stimulated humanCD4+ T cells. (A) Stimulation of human PBMCs in the presence of 100 μM3-HKyn and 100 μM 3-HAA, but not the downstream metabolites PA or QA,leads to the expansion of a population of cells expression the cytokineIL-22. (B) 3-HAA promotes IL-22 expression in dose-dependent manner butto a variable extent between individual donors. (C) IL-22 induction by3-HAA is blocked by inhibition of the AhR pathway. (D) Summary of 3different experiments with 6 donors demonstrating that inhibition of theAhR blocks IL-22 upregulation by 3-HAA.

FIG. 7: 3-HAA is not an AhR ligand. (A) Incubation of a murine hepatomacell line stably transfected with an AhR-responsive (AhR-eGFP) reporterconstruct in the presence of increasing concentrations of 3-HAA leads tominimal activation of the AhR. (B) Metabolic pathways downstream of3-HAA catalyzed by the enzyme 3-Hydroxyanthranilate 3,4-Dioxygenase(HAAO) including intermediates upstream of PA and QA generation. (C)HAAO blockade enhances the ability of 3-HAA to promote IL-22 expressionby stimulated CD4+ T cells. (D) Summary of two individuals showing thatHAAO inhibition increases the ability of 3-HAA to promote IL-22expression by CD4+ T cells in a dose-dependent manner.

FIG. 8: CA is an endogenous AhR agonist generated by oxidativedimerization of 3-HAA. (A) An alternative pathway of 3-HAA metabolismleads to the generation of CA either by non-enzymatic (oxidation) orenzymatic (laccase, ceruloplasmin) processes. CA is structurally similarto the synthetic AhR ligand TCDD. (B) CA acts as a potent agonist of theAhR, whereas 3-HAA has no activity in short-term assays. Tryptamine is apositive control. (C) Incubation of CA with in vitro translated humanAhR protein results in a dose-dependent inhibition of TCDD binding. (D)Incubation of sorted naïve human CD4+ T cells with CA (25 μM) potentlyinduces IL-22 whereas similar concentrations of 3-HAA only had minimaleffects. Data are representative of three similar experiments.

FIG. 9: The synthetic 3-HAA analogue 3,4 DAA (Tranilast) is an AhRantagonist and promotes the induction of Foxp3 by stimulated CD4+ Tcells. (A) 3,4 DAA shares some structural similarities to 3-HAA(highlighted region). (B) Stimulation of human PBMCs in the presence of3,4 DAA induces IL-22 expression by CD4+ T cells but to a lesser extentthan 3-HAA. (C) 3,4 DAA inhibits TCDD binding to human AhR in vitro butonly at high concentrations (1000 μM). (D) 3,4 DAA is blocks activationof the AhR by TCDD in H1G1 cells suggesting that it is an AhRantagonist. CH223191 (50 mM) is shown as a positive control. (E) 3,4 DAAinduces Foxp3 expression in stimulated naïve (CD45RA+CCR7+CD95−CD25−)CD4+ T cells. 3-HAA also promotes Foxp3 upregulation but to a lesserextent. The TGFβ signaling inhibitor SB-431542 (Activin-like kinase1inhibitor) reversed the ability of 3,4 DAA and 3-HAA to upregulateFoxp3, demonstrating that TGFβ is required for Foxp3 induction.

FIGS. 10A and 10B illustrate the number or frequency of cytokineproducing cells within the CNS (or spleen) of mice treated at day 0 with1 dose of CA (1 mg/mouse) or vehicle (DMSO) within the emulsion containMOG peptide and adjuvant. Representative flow plots are shown at thebottom of the figures. EAE survival curve for 10 animals treated with CAor with DMSO showing that 1/10 animals treated with CA gets sick whereas9/10 animals treated with vehicle (DMSO) are sick at the indicatedtimepoint.

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention provides compounds and methods for preventing andtreating an autoimmune disorder in a subject. The methods comprisingadministering to the subject compounds of the present invention in anamount sufficient prevent or treat an autoimmune disorder such asmultiple sclerosis, myasthenia gravis, Guillan-Barre syndrome(antiphospholipid syndrome), systemic lupus erytromatosis, Behcet'ssyndrome, Sjogrens syndrome, rheumatoid arthritis, Hashimoto'sdisease/hypothyroiditis, primary biliary cirrhosis, mixed connectivetissue disease, chronic active hepatitis, Graves'disease/hyperthyroiditis, scleroderma, chronic idiopathicthrombocytopenic purpura, diabetic neuropathy and septic shock. Themethods also comprise administering compounds of the present inventionto modulate AHR activity.

II. Definitions

As used herein, the term “alkyl” refers to a straight or branched,saturated, aliphatic radical having the number of carbon atomsindicated. For example, C₁-C₆ alkyl includes, but is not limited to,methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, pentyl, isopentyl, hexyl, etc. Other alkyl groups include,but are not limited to heptyl, octyl, nonyl, decyl, etc. Alkyl caninclude any number of carbons, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7,1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6 and 5-6. Thealkyl group is typically monovalent, but can be divalent, such as whenthe alkyl group links two moieties together.

The term “lower” referred to above and hereinafter in connection withorganic radicals or compounds respectively defines a compound or radicalwhich can be branched or unbranched with up to and including 7,preferably up to and including 4 and (as unbranched) one or two carbonatoms.

As used herein, the term “alkylene” refers to an alkyl group, as definedabove, linking at least two other groups, i.e., a divalent hydrocarbonradical. The two moieties linked to the alkylene can be linked to thesame atom or different atoms of the alkylene. For instance, a straightchain alkylene can be the bivalent radical of —(CH₂)_(n), where n is 1,2, 3, 4, 5 or 6. Alkylene groups include, but are not limited to,methylene, ethylene, propylene, isopropylene, butylene, isobutylene,sec-butylene, pentylene and hexylene.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′,—S(O)₂NR′R″, —CN and —NO₂ in a number ranging from zero to (2m′+1),where m′ is the total number of carbon atoms in such radical. R′, R″ andR′″ each independently refer to hydrogen, unsubstituted (C₁-C₈)alkyl andheteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens,unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C₁-C₄)alkylgroups. When R′ and R″ are attached to the same nitrogen atom, they canbe combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like). Preferably, thesubstituted alkyl and heteroalkyl groups have from 1 to 4 substituents,more preferably 1, 2 or 3 substituents. Exceptions are those perhaloalkyl groups (e.g., pentafluoroethyl and the like) which are alsopreferred and contemplated by the present invention.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

As used herein, the term “alkoxy” refers to alkyl group having an oxygenatom that either connects the alkoxy group to the point of attachment oris linked to two carbons of the alkoxy group. Alkoxy groups include, forexample, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy,iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. The alkoxygroups can be further substituted with a variety of substituentsdescribed within. For example, the alkoxy groups can be substituted withhalogens to form a “halo-alkoxy” group.

As used herein, the term “alkenyl” refers to either a straight chain orbranched hydrocarbon of 2 to 6 carbon atoms, having at least one doublebond. Examples of alkenyl groups include, but are not limited to, vinyl,propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl,1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl,1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl,1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkenyl groups canalso have from 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4to 6 and 5 to 6 carbons. The alkenyl groups is typically monovalent, butcan be divalent, such as when the alkenyl group links two moietiestogether.

As used herein, the term “alkenylene” refers to an alkenyl group, asdefined above, linking at least two other groups, i.e., a divalenthydrocarbon radical. The two moieties linked to the alkenylene can belinked to the same atom or different atoms of the alkenylene. Alkenylenegroups include, but are not limited to, ethenylene, propenylene,isopropenylene, butenylene, isobutenylene, sec-butenylene, pentenyleneand hexenylene.

As used herein, the term “alkynyl” refers to either a straight chain orbranched hydrocarbon of 2 to 6 carbon atoms, having at least one triplebond. Examples of alkynyl groups include, but are not limited to,acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl,butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl,1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl,1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl.Alkynyl groups can also have from 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to5, 3 to 6, 4 to 5, 4 to 6 and 5 to 6 carbons. The alkynyl groups istypically monovalent, but can be divalent, such as when the alkynylgroup links two moieties together.

As used herein, the term “alkynylene” refers to an alkynyl group, asdefined above, linking at least two other groups, i.e., a divalenthydrocarbon radical. The two moieties linked to the alkynylene can belinked to the same atom or different atoms of the alkynylene. Alkynylenegroups include, but are not limited to, ethynylene, propynylene,isopropynylene, butynylene, sec-butynylene, pentynylene and hexynylene.

As used herein, the term “alkyl amine” refers to an alkyl groups asdefined within, having one or more amino groups. The amino groups can beprimary, secondary or tertiary. The alkyl amine can be furthersubstituted with a hydroxy group. Alkyl amines useful in the presentinvention include, but are not limited to, ethyl amine, propyl amine,isopropyl amine, ethylene diamine and ethanolamine. The amino group canlink the alkyl amine to the point of attachment with the rest of thecompound, be at the omega position of the alkyl group, or link togetherat least two carbon atoms of the alkyl group. One of skill in the artwill appreciate that other alkyl amines are useful in the presentinvention.

As used herein, the term “halogen” refers to fluorine, chlorine, bromineand iodine.

As used herein, the term “haloalkyl” refers to alkyl as defined abovewhere some or all of the hydrogen atoms are substituted with halogenatoms. Halogen (halo) preferably represents chloro or fluoro, but mayalso be bromo or iodo. For example, haloalkyl includes trifluoromethyl,flouromethyl, 1,2,3,4,5-pentafluoro-phenyl, etc. The term “perfluoro”defines a compound or radical which has at least two available hydrogenssubstituted with fluorine. For example, perfluorophenyl refers to1,2,3,4,5-pentafluorophenyl, perfluoromethane refers to1,1,1-trifluoromethyl, and perfluoromethoxy refers to1,1,1-trifluoromethoxy.

As used herein, the term “halo-alkoxy” refers to an alkoxy group havingat least one halogen. Halo-alkoxy is as defined for alkoxy where some orall of the hydrogen atoms are substituted with halogen atoms. The alkoxygroups can be substituted with 1, 2, 3, or more halogens. When all thehydrogens are replaced with a halogen, for example by fluorine, thecompounds are per-substituted, for example, perfluorinated. Halo-alkoxyincludes, but is not limited to, trifluoromethoxy,2,2,2,-trifluoroethoxy, perfluoroethoxy, etc.

As used herein, the term “cycloalkyl” refers to a saturated or partiallyunsaturated, monocyclic, fused bicyclic or bridged polycyclic ringassembly containing from 3 to 12 ring atoms, or the number of atomsindicated Monocyclic rings include, for example, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Bicyclic andpolycyclic rings include, for example, norbornane, decahydronaphthaleneand adamantane. For example, C₃₋₈cycloalkyl includes cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbornane.

As used herein, the term “cycloalkylene” refers to a cycloalkyl group,as defined above, linking at least two other groups, i.e., a divalenthydrocarbon radical. The two moieties linked to the cycloalkylene can belinked to the same atom or different atoms of the cycloalkylene.Cycloalkylene groups include, but are not limited to, cyclopropylene,cyclobutylene, cyclopentylene, cyclohexylene, and cyclooctylene.

As used herein, the term “heterocycloalkyl” refers to a ring systemhaving from 3 ring members to about 20 ring members and from 1 to about5 heteroatoms such as N, O and S. Additional heteroatoms can also beuseful, including, but not limited to, B, Al, Si and P. The heteroatomscan also be oxidized, such as, but not limited to, —S(O)— and —S(O)₂—.For example, heterocycle includes, but is not limited to,tetrahydrofuranyl, tetrahydrothiophenyl, morpholino, pyrrolidinyl,pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, piperidinyl, indolinyl, quinuclidinyl and1,4-dioxa-8-aza-spiro[4.5]dec-8-yl.

As used herein, the term “heterocyclalkylene” refers to aheterocyclalkyl group, as defined above, linking at least two othergroups. The two moieties linked to the heterocyclalkylene can be linkedto the same atom or different atoms of the heterocyclalkylene.

As used herein, the term “aryl” refers to a monocyclic or fusedbicyclic, tricyclic or greater, aromatic ring assembly containing 6 to16 ring carbon atoms. For example, aryl may be phenyl, benzyl ornaphthyl, preferably phenyl. “Arylene” means a divalent radical derivedfrom an aryl group. Aryl groups can be mono-, di- or tri-substituted byone, two or three radicals selected from alkyl, alkoxy, aryl, hydroxy,halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy andoxy-C₂-C₃-alkylene; all of which are optionally further substituted, forinstance as hereinbefore defined; or 1- or 2-naphthyl; or 1- or2-phenanthrenyl. Alkylenedioxy is a divalent substitute attached to twoadjacent carbon atoms of phenyl, e.g. methylenedioxy or ethylenedioxy.Oxy-C₂-C₃-alkylene is also a divalent substituent attached to twoadjacent carbon atoms of phenyl, e.g. oxyethylene or oxypropylene. Anexample for oxy-C₂-C₃-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

Preferred as aryl is naphthyl, phenyl or phenyl mono- or disubstitutedby alkoxy, phenyl, halogen, alkyl or trifluoromethyl, especially phenylor phenyl-mono- or disubstituted by alkoxy, halogen or trifluoromethyl,and in particular phenyl.

Examples of substituted phenyl groups as R are, e.g. 4-chlorophen-1-yl,3,4-dichlorophen-1-yl, 4-methoxyphen-1-yl, 4-methylphen-1-yl,4-aminomethylphen-1-yl, 4-methoxyethylaminomethylphen-1-yl,4-hydroxyethylaminomethylphen-1-yl,4-hydroxyethyl-(methyl)-aminomethylphen-1-yl, 3-aminomethylphen-1-yl,4-N-acetylaminomethylphen-1-yl, 4-aminophen-1-yl, 3-aminophen-1-yl,2-aminophen-1-yl, 4-phenyl-phen-1-yl, 4-(imidazol-1-yl)-phen-yl,4-(imidazol-1-ylmethyl)-phen-1-yl, 4-(morpholin-1-yl)-phen-1-yl,4-(morpholin-1-ylmethyl)-phen-1-yl,4-(2-methoxyethylaminomethyl)-phen-1-yl and4-(pyrrolidin-1-ylmethyl)-phen-1-yl, 4-(thiophenyl)-phen-1-yl,4-(3-thiophenyl)-phen-1-yl, 4-(4-methylpiperazin-1-yl)-phen-1-yl, and4-(piperidinyl)-phenyl and 4-(pyridinyl)-phenyl optionally substitutedin the heterocyclic ring.

As used herein, the term “arylene” refers to an aryl group, as definedabove, linking at least two other groups. The two moieties linked to thearylene are linked to different atoms of the arylene. Arylene groupsinclude, but are not limited to, phenylene.

As used herein, the term “arylene-oxy” refers to an arylene group, asdefined above, where one of the moieties linked to the arylene is linkedthrough an oxygen atom. Arylene-oxy groups include, but are not limitedto, phenylene-oxy.

Similarly, substituents for the aryl and heteroaryl groups are variedand are selected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN,—NO₂, —CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)₂R′,—NR′—C(O)NR″R′″, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —N₃, —CH(Ph)₂, perfluoro(C₁-C₄)alkoxy, andperfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total numberof open valences on the aromatic ring system; and where R′, R″ and R′″are independently selected from hydrogen, (C₁-C₈)alkyl and heteroalkyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl,and (unsubstituted aryl)oxy-(C₁-C₄)alkyl.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CH₂)_(q)—U—, wherein T and U are independently —NH—, —O—, —CH₂—or a single bond, and q is an integer of from 0 to 2. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula-A-(CH₂)_(r)—B—, wherein A and B are independently —CH₂—, —O—, —NH—,—S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integerof from 1 to 3. One of the single bonds of the new ring so formed mayoptionally be replaced with a double bond. Alternatively, two of thesubstituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CH₂)_(s)—X—(CH₂)_(t)—, where s and t are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituent R′ in —NR′— and —S(O)₂NR′— is selected from hydrogen orunsubstituted (C₁-C₆)alkyl.

As used herein, the term “alkyl-aryl” refers to a radical having analkyl component and an aryl component, where the alkyl component linksthe aryl component to the point of attachment. The alkyl component is asdefined above, except that the alkyl component is at least divalent inorder to link to the aryl component and to the point of attachment. Insome instances, the alkyl component can be absent. The aryl component isas defined above. Examples of alkyl-aryl groups include, but are notlimited to, benzyl.

As used herein, the term “alkenyl-aryl” refers to a radical having bothan alkenyl component and a aryl component, where the alkenyl componentlinks the aryl component to the point of attachment. The alkenylcomponent is as defined above, except that the alkenyl component is atleast divalent in order to link to the aryl component and to the pointof attachment. The aryl component is as defined above. Examples ofalkenyl-aryl include ethenyl-phenyl, among others.

As used herein, the term “Heteroaryl” refers to a monocyclic or fusedbicyclic or tricyclic aromatic ring assembly containing 5 to 16 ringatoms, where from 1 to 4 of the ring atoms are a heteroatom each N, O orS. For example, heteroaryl includes pyridyl, indolyl, indazolyl,quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl,furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl,triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any otherradicals substituted, especially mono- or di-substituted, by e.g. alkyl,nitro or halogen. Pyridyl represents 2-, 3- or 4-pyridyl, advantageously2- or 3-pyridyl. Thienyl represents 2- or 3-thienyl. Quinolinylrepresents preferably 2-, 3- or 4-quinolinyl. Isoquinolinyl representspreferably 1-, 3- or 4-isoquinolinyl. Benzopyranyl, benzothiopyranylrepresents preferably 3-benzopyranyl or 3-benzothiopyranyl,respectively. Thiazolyl represents preferably 2- or 4-thiazolyl, andmost preferred, 4-thiazolyl. Triazolyl is preferably 1-, 2- or5-(1,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl.

Preferably, heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl,thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl,thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl,benzothienyl, oxazolyl, indazolyl, or any of the radicals substituted,especially mono- or di-substituted.

As used herein, the term “salt” refers to acid or base salts of thecompounds used in the methods of the present invention. Illustrativeexamples of pharmaceutically acceptable salts are mineral acid(hydrochloric acid, hydrobromic acid, phosphoric acid, and the like)salts, organic acid (acetic acid, propionic acid, glutamic acid, citricacid and the like) salts, quaternary ammonium (methyl iodide, ethyliodide, and the like) salts. It is understood that the pharmaceuticallyacceptable salts are non-toxic. Additional information on suitablepharmaceutically acceptable salts can be found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, which is incorporated herein by reference.

As used herein, the term “immune disorder” refers to a dysfunction ofthe immune system. An immune disorder can be characterized in severalways, including certain components involved in the immune system,whether the immune system is overactive or underactive, or whether thedisorder is congenital or acquired. An immune disorder can include anautoimmune disorder including multiple sclerosis, myasthenia gravis,Guillan-Barre syndrome (antiphospholipid syndrome), systemic lupuserytromatosis, Behcet's syndrome, Sjogrens syndrome, rheumatoidarthritis, Hashimoto's disease/hypothyroiditis, primary biliarycirrhosis, mixed connective tissue disease, chronic active hepatitis,Graves' disease/hyperthyroiditis, scleroderma, chronic idiopathicthrombocytopenic purpura, diabetic neuropathy and septic shock. Animmune disorder can further include immunodeficiency disorders andallergies.

“Subject” refers to animals such as mammals, including, but not limitedto, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats,rabbits, rats, mice and the like. In certain embodiments, the subject isa human.

As used herein, the terms “therapeutically effective amount or dose” or“therapeutically sufficient amount or dose” or “effective or sufficientamount or dose” refer to a dose that produces therapeutic effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, thetherapeutically effective dose can often be lower than the conventionaltherapeutically effective dose for non-sensitized cells.

As used herein, the term “pharmaceutically acceptable excipient” refersto a substance that aids the administration of an active agent to andabsorption by a subject. Pharmaceutical excipients useful in the presentinvention include, but are not limited to, binders, fillers,disintegrants, lubricants, coatings, sweeteners, flavors and colors. Oneof skill in the art will recognize that other pharmaceutical excipientsare useful in the present invention.

As used herein, the terms “administer” or “administering” refer to oraladministration, administration as a suppository, topical contact,parenteral, intravenous, intraperitoneal, intramuscular, intralesional,intranasal or subcutaneous administration, intrathecal administration,or the implantation of a slow-release device e.g., a mini-osmotic pump,to the subject.

As used herein, the terms “treat” or “treating” or “treatment” refers toany indicia of success in the treatment or amelioration of an injury,pathology, condition, or symptom (e.g., pain), including any objectiveor subjective parameter such as abatement; remission; diminishing ofsymptoms or making the symptom, injury, pathology or condition moretolerable to the patient; decreasing the frequency or duration of thesymptom or condition; or, in some situations, preventing the onset ofthe symptom or condition. The treatment or amelioration of symptoms canbe based on any objective or subjective parameter; including, e.g., theresult of a physical examination.

As used herein, the terms “patient” or “patient in need” refers to asubject suffering from an autoimmune disorder including multiplesclerosis, myasthenia gravis, Guillan-Barre syndrome (antiphospholipidsyndrome), systemic lupus erytromatosis, Behcet's syndrome, Sjogrenssyndrome, rheumatoid arthritis, Hashimoto's disease/hypothyroiditis,primary biliary cirrhosis, mixed connective tissue disease, chronicactive hepatitis, Graves' disease/hyperthyroiditis, scleroderma, chronicidiopathic thrombocytopenic purpura, diabetic neuropathy and septicshock. Patients suffering from other conditions treatable with thedisclosed compounds are also treatable with the methods of the presentinvention. Patients treatable using the methods of the present inventionare animals such as mammals, including, but not limited to, primates(e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats,mice and the like. In certain embodiments, the patient is a human.

III. Compounds

The present invention provides compounds for treating autoimmunedisorders.

In some embodiments, the present invention provides compounds of formulaIa:

wherein each of R^(1a), R^(1b), and R^(1c) is independently selectedfrom the group consisting of hydrogen, alkyl, substituted alkyl,alkylene, substituted alkylene, heteroalkyl, substituted heteroalkyl,alkoxy, alkenyl, alkenylene, alkynyl, alkynylene, amine, alkyl amine,halogen, haloalkyl, halo-alkoxy, cycloakyl, cycloalkylene,heterocycloalkyl, heterocycloalkylene, aryl, arylene, arylene-oxy,alkyl-aryl, alkenyl-aryl, and heteroaryl, and each of R^(2a), R^(2b),R^(2c), and R^(2d) is independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, alkylene, substituted alkylene,heteroalkyl, substituted heteroalkyl, alkoxy, alkenyl, alkenylene,alkynyl, alkynylene, amine, alkyl amine, halogen, haloalkyl,halo-alkoxy, cycloakyl, cycloalkylene, heterocycloalkyl,heterocycloalkylene, aryl, arylene, arylene-oxy, alkyl-aryl,alkenyl-aryl, and heteroaryl. With the proviso that when R^(1b) is —NH₂,then R^(1c) or R^(2d) is not —CO₂H. With the proviso that when R^(1c)and R^(2d) are —CO₂H, then R^(1b) is not —NH₂. The compounds of formula1a include salts and isomers thereof.

In some other embodiments, the present invention provides compounds offormula Ib:

wherein each R¹ is independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, alkylene, substituted alkylene,heteroalkyl, substituted heteroalkyl, alkoxy, alkenyl, alkenylene,alkynyl, alkynylene, amine, alkyl amine, halogen, haloalkyl,halo-alkoxy, cycloakyl, cycloalkylene, heterocycloalkyl,heterocycloalkylene, aryl, arylene, arylene-oxy, alkyl-aryl,alkenyl-aryl, and heteroaryl, and each R² is independently selected fromthe group consisting of hydrogen, hydrogen, alkyl, substituted alkyl,alkylene, substituted alkylene, heteroalkyl, substituted heteroalkyl,alkoxy, alkenyl, alkenylene, alkynyl, alkynylene, amine, alkyl amine,halogen, haloalkyl, halo-alkoxy, cycloakyl, cycloalkylene,heterocycloalkyl, heterocycloalkylene, aryl, arylene, arylene-oxy,alkyl-aryl, alkenyl-aryl, and heteroaryl. Subscript m of formula 1b isan integer from 1 to 3, and subscript n is an integer from 1 to 4. Thecompounds of formula 1b include salts and isomers thereof.

In yet some other embodiments, the compound of formula Ib is:

Pharmaceutically acceptable salts of the acidic compounds of the presentinvention are salts formed with bases, namely cationic salts such asalkali and alkaline earth metal salts, such as sodium, lithium,potassium, calcium, magnesium, as well as ammonium salts, such asammonium, trimethyl-ammonium, diethylammonium, andtris-(hydroxymethyl)-methyl-ammonium salts.

Similarly acid addition salts, such as of mineral acids, organiccarboxylic and organic sulfonic acids, e.g., hydrochloric acid,methanesulfonic acid, maleic acid, are also possible provided a basicgroup, such as pyridyl, constitutes part of the structure.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are all intended to beencompassed within the scope of the present invention.

The compounds of the present invention can be prepared by a variety ofmethods known to one of skill in the art, for example, see Richard C.Larock, Comprehensive Organic Transformations 1989, VCH Publishers, Inc.

IV. Compositions and Administration

The present invention also provides compositions of a compound of thepresent invention and a pharmaceutically acceptable excipient. In someembodiments, the compound of the present invention is a compound offormula Ia or Ib. Pharmaceutical excipients useful in the presentinvention include, but are not limited to, binders, fillers,disintegrants, lubricants, coatings, sweeteners, flavors and colors. Oneof skill in the art will recognize that other pharmaceutical excipientsare useful in the present invention.

The compounds of the present invention can be administered as frequentlyas necessary, including hourly, daily, weekly or monthly. The compoundsutilized in the pharmaceutical method of the invention are administeredat the initial dosage of about 0.0001 mg/kg to about 1000 mg/kg daily. Adaily dose range of about 0.01 mg/kg to about 500 mg/kg, or about 0.1mg/kg to about 200 mg/kg, or about 1 mg/kg to about 100 mg/kg, or about10 mg/kg to about 50 mg/kg, can be used. The dosages, however, may bevaried depending upon the requirements of the patient, the severity ofthe condition being treated, and the compound being employed. Forexample, dosages can be empirically determined considering the type andstage of disease diagnosed in a particular patient. The doseadministered to a patient, in the context of the present inventionshould be sufficient to effect a beneficial therapeutic response in thepatient over time. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side-effects that accompanythe administration of a particular compound in a particular patient.Determination of the proper dosage for a particular situation is withinthe skill of the practitioner. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under circumstances is reached. For convenience, thetotal daily dosage may be divided and administered in portions duringthe day, if desired. Doses can be given daily, or on alternate days, asdetermined by the treating physician. Doses can also be given on aregular or continuous basis over longer periods of time (weeks, monthsor years), such as through the use of a subdermal capsule, sachet ordepot, or via a patch.

The pharmaceutical compositions can be administered to the patient in avariety of ways, including topically, parenterally, intravenously,intradermally, intramuscularly, colonically, rectally orintraperitoneally. Preferably, the pharmaceutical compositions areadministered parenterally, topically, intravenously, intramuscularly ororally.

The compounds of the present invention can be administered to a subjectusing any suitable methods known in the art. For example, a compound offormula Ia or Ib can be formulated as pharmaceutical compositions with apharmaceutically acceptable diluent, carrier or excipient. Suitableformulations for use in the present invention are found in Remington'sPharmaceutical Sciences (17th ed. 1985)), which is incorporated hereinby reference. A brief review of methods for drug delivery is alsodescribed in, e.g., Langer, Science 249:1527-1533 (1990), which isincorporated herein by reference.

A compound of formula Ia or Ib can be administered in anypharmaceutically acceptable composition. A pharmaceutically acceptablenontoxic composition is formed by incorporating any of normally employedexcipients, and generally 10-95% of active ingredient and morepreferably at a concentration of 25%-75%. Furthermore, to improve oralabsorption of a compound of formula Ia or Ib, various carrier systems,such as nanoparticles, microparticles, liposomes, phospholipids,emulsions, erythrocytes, etc. can be used. The oral agents comprising acompound of formula Ia or Ib of the invention can be in any suitableform for oral administration, such as liquid, tablets, capsules, or thelike. The oral formulations can be further coated or treated to preventor reduce dissolution in stomach.

Furthermore, a compound of formula Ia or Ib can be formulated forparenteral, topical, nasal, sublingual, gavage, or local administration.For example, the pharmaceutical compositions are administeredparenterally, e.g., intravenously, subcutaneously, intradermally, orintramuscularly, or intranasally. Thus, the invention providescompositions for parenteral administration that comprise a solution of asingle or mixture of a compound of formula Ia or Ib, dissolved orsuspended in an acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers may be used including, for example, water,buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.These compositions may be sterilized by conventional, well knownsterilization techniques, or they may be sterile filtered. The resultingaqueous solutions may be packaged for use as is or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionsincluding pH adjusting and buffering agents, tonicity adjusting agents,wetting agents and the like, such as, for example, sodium acetate,sodium lactate, sodium chloride, potassium chloride, calcium chloride,sorbitan monolaurate, triethanolamine oleate, etc.

For aerosol administration, a compound of formula Ia or Ib is preferablysupplied in finely divided form along with a surfactant and propellant.The surfactant must, of course, be nontoxic, and preferably soluble inthe propellant. Representative of such agents are the esters or partialesters of fatty acids containing from 6 to 22 carbon atoms, such ascaproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,olesteric and oleic acids with an aliphatic polyhydric alcohol or itscyclic anhydride. Mixed esters, such as mixed or natural glycerides maybe employed. A carrier can also be included, as desired, as with, e.g.,lecithin for intranasal delivery.

For solid compositions, conventional nontoxic solid carriers may beused. Solid carriers include, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like.

Compounds of the present invention can also be introduced into an animalcell, preferably a mammalian cell, via a liposomes and liposomederivatives such as immunoliposomes and lipid:nucleic acid complexes.The term “liposome” refers to vesicles comprised of one or moreconcentrically ordered lipid bilayers, which encapsulate an aqueousphase. The aqueous phase typically contains the compound to be deliveredto the cell, i.e., a compound of formula 1a and 1b.

The liposome fuses with the plasma membrane, thereby releasing the acompound of formula 1a or 1b into the cytosol. Alternatively, theliposome is phagocytosed or taken up by the cell in a transport vesicle.Once in the endosome or phagosome, the liposome either degrades or fuseswith the membrane of the transport vesicle and releases its contents.

In current methods of drug delivery via liposomes, the liposomeultimately becomes permeable and releases the encapsulated compound (inthis case, a compound of formula Ia or Ib) at the target tissue or cell.For systemic or tissue specific delivery, this can be accomplished, forexample, in a passive manner wherein the liposome bilayer degrades overtime through the action of various agents in the body. Alternatively,active drug release involves using an agent to induce a permeabilitychange in the liposome vesicle. Liposome membranes can be constructed sothat they become destabilized when the environment becomes acidic nearthe liposome membrane (see, e.g., Proc. Nat'l Acad. Sci. USA 84:7851(1987); Biochemistry 28:908 (1989)). When liposomes are endocytosed by atarget cell, for example, they become destabilized and release theircontents. This destabilization is termed fusogenesis.Dioleoylphosphatidylethanolamine (DOPE) is the basis of many “fusogenic”systems.

Such liposomes typically comprise a compound of formula Ia or Ib and alipid component, e.g., a neutral and/or cationic lipid, optionallyincluding a receptor-recognition molecule such as an antibody that bindsto a predetermined cell surface receptor or ligand (e.g., an antigen). Avariety of methods are available for preparing liposomes as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S.Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054,4,501,728, 4,774,085, 4,837,028, 4,235,871, 4,261,975, 4,485,054,4,501,728, 4,774,085, 4,837,028, 4,946,787, PCT Publication No. WO91/17424, Deamer & Bangham, Biochim. Biophys. Acta 443:629-634 (1976);Fraley, et al., Proc. Nat'l Acad. Sci. USA 76:3348-3352 (1979); Hope ctal., Biochim. Biophys. Acta 812:55-65 (1985); Mayer et al., Biochim.Biophys. Acta 858:161-168 (1986); Williams et al., Proc. Nat'l Acad.Sci. USA 85:242-246 (1988); Liposomes (Ostro (ed.), 1983, Chapter 1);Hope et al., Chem. Phys. Lip. 40:89 (1986); Gregoriadis, LiposomeTechnology (1984) and Lasic, Liposomes: from Physics to Applications(1993)). Suitable methods include, for example, sonication, extrusion,high pressure/homogenization, microfluidization, detergent dialysis,calcium-induced fusion of small liposome vesicles and ether-fusionmethods, all of which are well known in the art.

In certain embodiments of the present invention, it is desirable totarget the liposomes of the invention using targeting moieties that arespecific to a particular cell type, tissue, and the like. Targeting ofliposomes using a variety of targeting moieties (e.g., ligands,receptors, and monoclonal antibodies) has been previously described(see, e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044). Standard methodsfor coupling targeting agents to liposomes can be used. These methodsgenerally involve incorporation into liposomes lipid components, e.g.,phosphatidylethanolamine, which can be activated for attachment oftargeting agents, or derivatized lipophilic compounds, such as lipidderivatized bleomycin. Antibody targeted liposomes can be constructedusing, for instance, liposomes which incorporate protein A (seeRenneisen et al., J. Biol. Chem., 265:16337-16342 (1990) and Leonetti etal., Proc. Nat'l Acad. Sci. USA 87:2448-2451 (1990).

In some therapeutic applications, a compound of formula 1a or 1b of theinvention is administered to a patient in an amount sufficient todecrease symptoms of an autoimmune disorder. An amount adequate toaccomplish this is defined as “therapeutically effective dose.” Amountseffective for this use will depend on, for example, the particularcompound of formula Ia or Ib employed, the manner of administration, theweight and general state of health of the patient, and the judgment ofthe prescribing physician. “Therapeutically effective dose” alsoencompasses doses that are sufficient to prevent an autoimmune diseasefrom developing in a subject. Thus, prophylactic doses are encompassedby the term “therapeutically effective dose.” The foregoing are generalguidelines only that can be expanded or altered based on, for example,disease type and grade, patient age, health status, and sex, theparticular drugs used in combination, the route and frequency ofadministration, and experimental and clinical findings using a multidrugcombination.

V. Methods of Treating Immune Disorders

In some embodiments, the present invention provides methods forpreventing and treating an autoimmune disorder in a subject. The methodcomprises administering to the subject a compound of formula 1a or 1b inan amount sufficient prevent or treat an autoimmune disorder such asmultiple sclerosis, myasthenia gravis, Guillan-Barre syndrome(antiphospholipid syndrome), systemic lupus crytromatosis, Behcet'ssyndrome, Sjogrens syndrome, rheumatoid arthritis, Hashimoto'sdisease/hypothyroiditis, primary biliary cirrhosis, mixed connectivetissue disease, chronic active hepatitis, Graves'disease/hyperthyroiditis, scleroderma, chronic idiopathicthrombocytopenic purpura, diabetic neuropathy and septic shock.

In another aspect, the present invention provides a method of modulatingAryl Hydrocarbon Receptor (AHR) activity for treating and/or preventingautoimmune disorders. In some embodiments the method includes contactinga compound of formula 1a or 1b with the AHR. In some embodiments themethod includes contacting a compound of formula Ia or 1b or apharmaceutical composition thereof, with the AHR.

Treatment methods provided herein include, in general, administration toa patient an effective amount of one or more compounds provided herein,e.g., orally, nasally or parenterally. Suitable patients include thosepatients suffering from or susceptible to (i.e., prophylactic treatment)a disorder or disease identified herein. Typical patients for treatmentas described herein include mammals, particularly primates, especiallyhumans. Other suitable patients include domesticated companion animalssuch as a dog, cat, horse, and the like, or a livestock animal such ascattle, pig, sheep and the like.

In general, treatment methods provided herein comprise administering toa patient an effective amount of a compound of formula 1a or 1b. In oneembodiment, the compound(s) of the invention are administered to apatient (e.g., a human) orally. The effective amount may be an amountsufficient to modulate the AHR receptor and/or an amount sufficient toreduce or alleviate the symptoms presented by the patient. Treatmentregimens may vary depending on the compound used and the particularcondition to be treated. The compounds of the present invention can beadministered as frequently as necessary, including hourly, daily, weeklyor monthly. It will be understood, however, that the specific dose leveland treatment regimen for any particular patient will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex, diet, time ofadministration, route of administration, rate of excretion, drugcombination (i.e., other drugs being administered to the patient) andthe severity of the particular disease undergoing therapy, as well asthe judgment of the prescribing medical practitioner. In general, theuse of the minimum dose sufficient to provide effective therapy ispreferred. Patients may generally be monitored for therapeuticeffectiveness using medical or veterinary criteria suitable for thecondition being treated or prevented. Generally, treatment is initiatedwith smaller dosages which are less than the optimum dose of thecompound. Thereafter, the dosage is increased by small increments untilthe optimum effect under circumstances is reached. For convenience, thetotal daily dosage may be divided and administered in portions duringthe day, if desired. Doses can be given daily, or on alternate days, asdetermined by the treating physician. Doses can also be given on aregular or continuous basis over longer periods of time (weeks, monthsor years), such as through the use of a subdermal capsule, sachet ordepot, or via a patch.

The pharmaceutical compositions can be administered to the patient in avariety of ways, including topically, parenterally, intravenously,intradermally, intramuscularly, colonically, rectally orintraperitoneally. Preferably, the pharmaceutical compositions areadministered parenterally, topically, intravenously, intramuscularly ororally.

VI. Examples

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

FIG. 1 shows that Cinnabarinic Acid inhibits the development ofexperimental allergic encephalomyelitis (EAE) in C57BL/6 mice with asingle dose given at the time that MOG peptide and CFA were injected—Day0

Example 2

FIG. 2 shows that Cinnabarinic Acid activates AHR responsive (DioxinResponsive Elements) in an in vitro test demonstrating agonist activity.Tryptamine is a positive control and a downstream related molecule(Compound 1) is a negative control

Example 3

FIG. 3 shows that Cinnabarinic Acid displaces Dioxin (TCDD) in an invitro assay using purified in vitro translated human AHR protein therebydemonstrating direct binding

Example 4

FIG. 4 shows that Cinnabarinic Acid potently induces IL-22 in vitro incultured human naïve CD4+ T cells stimulated with anti-CD3/anti-CD28under “Th17” polarizing conditions (IL-1b, IL-21, IL-23 with anti-IFNgand anti-IL4). This is AHR dependent as inclusion of an AHR antagonistfully abrogated effect.

Example 5

FIG. 5 shows that Cinnabaric Acid inhibits the induction of EAE ascompared to DMSO.

Example 6

The aryl hydrocarbon receptor (AHR) binds to environmental toxins andsynthetic aromatic halogenated hydrocarbons and is involved in a diversearray of biological processes. Recently the AHR was shown to controlhost immunity by affecting the balance between inflammatory T cells thatproduce interleukin 17 (Th17) and regulatory T cells (Treg) that areinvolved in tolerance. While environmental toxins were shown to mediatethis effect, suggesting a link between pollutants and autoimmunity, itis likely that natural ligands also exist which may play an importantrole in regulating host immune responses in a similar manner. Weinvestigated downstream metabolites of tryptophan as potential AHRligands because (1) tryptophan metabolites have been implicated inregulating the balance between Th17 and Treg cells and (2) many of theAHR ligands identified thus far are derivatives of tryptophan. Here weidentify cinnabarinic acid (CA) as an AHR ligand downstream ofinflammatory tryptophan metabolism. CA was found to stimulate thedifferentiation of a newly defined subset of human T cells that producethe cytokine IL-22, but not IL-17, and suppress autoimmunity in a mousemodel of multiple sclerosis. Our findings link inflammatory tryptophanmetabolism to activation of the AHR in humans and mice and define anovel endogenous ligand of the AHR that may have broad biologicalfunctions.

The enzyme indole 2,3-dioxygenase (IDO) plays an important role in theregulation of both the innate and adaptive immune system in settingssuch as cancer, autoimmunity, microbial pathogenesis, and pregnancy(1-4). IDO is the first, rate-limiting step in the metabolism of theessential amino acid tryptophan to kynurenine and is induced undercertain inflammatory conditions, most notably in response to interferons(IFN) (reviewed in 5). Two non-exclusive mechanisms have been suggestedfor how IDO affects immunity: (a) through depleting local tryptophanlevels leading to the induction of the ‘amino acid starvation’ response(6), and (b) through the generation of kynurenine and other downstreammetabolites which have specific immunomodulatory or cytotoxic functions(7). Tryptophan metabolites generated through the IDO pathway have beenshown to suppress T cell activation and to affect T cell differentiationpathways, although the means by which they achieve these effects remainlargely unknown (8-10). A potential mechanism that was recentlyidentified suggests that tryptophan metabolites can alter the balance ofTreg cells and Th17 cells, two related populations of T cells that haveopposing functions during immune responses (11).

Treg and Th17 cells share similar developmental pathways and may arisefrom a common progenitor population (12). Several different mechanismshave been identified that govern the decision of a T cell to become aTreg or Th17 cell including the presence of inflammatory cytokines(IL-1b, IL-6, IL-21, IL-23) (13), retinoic acid (14), and activation ofthe AHR (15,16). The AHR is a cytosolic transcription factor that isinvolved in many biological processes including development, cellulardifferentiation and proliferation, metabolism of xenobiotic agents, andthe immune response (reviewed in 15). The best-studied ligands to dateare synthetic halogenated and polycyclic aromatic hydrocarbons (mostnotably 2,3,7,8-tetrachlorodibenzodioxin, (TCDD)) (17). Only a fewnatural endogenous ligands have been identified, many of which aretryptophan derivatives. These include2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester(ITE), tryptamine, indigo and indirubin, and6-formylindolo[3,2-b]carbazole (FICZ) (17). The highly conserved natureof the AHR signaling pathway has prompted the search for additionalendogenous ligands that can be directly linked to physiologicalfunctions. We therefore sought to determine whether tryptophanmetabolites downstream of IDO might act as AHR ligands.

3-Hydroxyanthranilic Acid (3-HAA) Induces IL-22 Expression by ActivatedCD4+ T Cells

Although the AHR was initially proposed to impact Treg and Th17development, more recent findings suggest that the expression of thecytokine IL-22, which is often co-expressed by Th17 cells in mice, ismore greatly impacted by AHR activation (16). This is highlighted by theobservation that AHR−/− mice retain the ability to generate some Th17cells, but are incapable of generating cells that express IL-22 (16-18).Human T cells were also found to exhibit distinct requirements for theAHR in T cell differentiation. Activation of the AHR in stimulated humanT cells by FICZ was found to have inhibitory effects, if any, on Th17differentiation, but promoted a significant increase in the frequency ofCD4+ T cells that produced IL-22 (Th22) (19). To screen for the abilityof tryptophan metabolites generated downstream of IDO to activate theAHR, we stimulated human T cells in vitro in the presence of differenttryptophan metabolites (3-Hydroxykynurenine (3-HKA),3-Hydroxyanthranilic Acid (3-HAA), Picolinic Acid (PA), and QuinolinicAcid (QA)) to assess whether any promoted the expansion of CD4+ T cellssecreting IL-22. We found that 3-HKA and 3-HAA, but not the downstreammetabolites PA or QA, were able to promote the differentiation of Th22cells (FIG. 6A). These cells frequently co-expressed IFNγ but weregenerally found to be IL-17A negative, suggesting a phenotype comparableto the Th22 cells recently identified in humans (19). We also found thatdifferent donors induced different levels of IL-22 upon activationalthough in each case 3-HAA was able to promote at least a 2-foldexpansion of Th22 cells (FIG. 6B). To determine whether the induction ofIL-22 within this population was dependent upon AHR activation, we nextstimulated human T cells in the presence or absence of a potent AHRantagonist (CH-223191) and monitored the induction of IL-22. We observedthat CH-223191 was able to significantly abolish IL-22 induction by3-HAA, suggesting that the AHR was required for this effect (FIG. 6C,D).

3-HKA and 3-HAA are not AHR Ligands but May be Precursors to an AHRLigand

Our initial screen examining AHR-dependent induction of IL-22 by humanCD4+ T cells indicated that 3-HAA, and to a lesser extent 3-HKA, werepotential ligands of the AHR. Because 3-HKA is a precursor to 3-HAA, wehypothesized that the ability of 3-HKA to promote IL-22 inductionresulted from the generation of 3-HAA in our culture conditions, andthat 3-HAA was the most likely candidate as an AHR ligand. We performeda precursory screen of all tryptophan metabolites including both 3-HKAand 3-HAA to bind to and act as agonists or antagonists of the AHR usingtwo separate assays. First we examined the ability of 3-HKyn, 3-HAA, andPA to displace TCDD binding to the human AHR protein in vitro (20). Wewere able to detect modest binding of 3-HAA only at very highconcentrations, and were unable to measure any significant displacementusing any of the other tryptophan metabolites tested. We also measuredAHR activation using a mouse hepatoma cell line (H1G1) that stablyexpresses a green fluorescent protein (GFP) reporter constructdownstream of a series of dioxin responsive elements (FIG. 7A) (21). Wemeasured both agonistic, and antagonistic, activity of each metabolitein addition to known agonists (Tryptamine, FICZ) and antagonists(CH-223191). While we noted a modest increase in AHR activity using3-HAA as an agonist, this failed to reach significance, and none of theother metabolites (3-HKyn, PA, QA) were found to have any activity asagonists or antagonists. These results suggested that 3-HAA might be anupstream precursor of an AHR ligand.

PA and QA are generally regarded as the primary metabolites generateddownstream of 3-HAA during tryptophan metabolism through the IDOpathway. As neither of these was found to have any impact on IL-22induction in our initial screen, we thought it would be unlikely thateither was an AHR ligand. The enzyme involved upstream of the generationof PA and QA is called 3-Hydroxyanthranilate 3,4-Dioxygenase (HAAO) andis expressed by the same cell populations that express IDO underinflammatory conditions (22). HAAO converts 3-HAA to2-amino-3-carboxymuconic semialdehyde, which may be non-enzymaticallycyclized to form QA or can be enzymatically converted into2-amino-muconic-semialdehyde, which in turn cyclizes to form PA (23).2-amino-3-carboxymuconic semialdehyde can also be further metabolized toform Acetyl-CoA (FIG. 7B) (23). To address whether any downstreamintermediates of 3-HAA generated through the activity of the enzyme HAAOwere acting as AHR ligands, we utilized the specific HAAO inhibitor4-fluoro-3-hydroxythranilic acid (4-F-3-HAA), in the presence ofdifferent concentrations of 3-HAA, to prevent the generation ofdownstream metabolites. Surprisingly we observed a stark increase in theability of 3-HAA to induce IL-22 under our culture conditions (FIG. 7C,D). This finding suggests that alternative pathways are likely to existwhich might be involved in the generation of AHR ligands downstream of3-HAA.

Identification of Cinnabarinic Acid as an AHR Ligand Downstream of 3-HAA

3-HAA is particularly susceptible to oxidation, resulting in theformation of the metabolite cinnabarinic acid (CA) through dimerizationof two molecules of 3-HAA (24). As a polycyclic aromatic hydrocarbon,cinnabarinic acid's structural features suggest it may act as an AHRligand (FIG. 8A). In addition, CA has potent effects on thymocytematuration (25) not unlike what has been observed in rodents treatedwith TCDD (26-28) or in transgenic mice with a constitutively active AHR(29). To test the hypothesis that CA is an AHR ligand, we obtained pureCA, prepared through a chemical synthesis using 3-HAA as a startingmaterial and purified by HPLC, and analyzed its ability to bind to andactivate the AHR in vitro. We found that CA was able to bind to andactivate the AHR at much lower concentrations than that observed for3-HAA (FIG. 8B), resulting in upregulation of CYP1A1 transcriptionwithin lymphocytes. The ability of CA to serve as an AHR ligand isconserved across species, as measured by CA's potent induction of CYP1A1in zebrafish embryos within 72 hours. Consequently, treatment ofactivated human naïve CD4+ T cells with CA promoted IL-22 productionmore strongly than any other tryptophan metabolite tested (FIG. 8C).

The 3-HAA Synthetic Analogue Tranilast is an Antagonist of the AHR

N-(3,4-dimethoxycinnamonyl) anthranilic acid (3,4-DAA; Tranilast) isthought to have immunosuppressive properties similar to 3-HAA, yet itsprecise mode of action in suppressing immune responses remains unclear(25). 3,4 DAA shares the anthranilate core with 3-HAA, yet based on itsstructure, it is unlikely to dimerize into a polycyclic ring system suchas that of CA (FIG. 9A). We found that 3,4 DAA promoted a slightincrease in the frequency of CD4+ T cells secreting IL-22 in our invitro stimulations with human T cells (FIG. 9B); in contrast to 3-HAA,3,4 DAA also dramatically suppressed T cell proliferation in the rangeof concentrations that were tested. 3,4 DAA was able to bind to thehuman AHR in vitro in our cell-free system and displace TCDD atrelatively high concentrations, similar to those seen for 3-HAA (FIG.9C), and it had no activity as an AHR agonist in our reporter assays.Co-treatment of our H1G1AHR reporter cells with 3,4 DAA prevented AHRactivation by TCDD, indicating 3,4 DAA actually may serve as an AHRantagonist (*NB we could say antagonist of AHR transcriptionalactivities . . . ), albeit to a lesser extent than the potent antagonistCH-223191 (FIG. 9D).

In a previous report where 3,4 DAA was used to suppress the developmentof EAE, it was noted that T cells from treated mice were able tosuppress EAE upon transfer into wild-type mice, suggesting that themechanism underlying suppression involved the development of suppressiveT cell subsets (25). Therefore we asked whether T cells treated with 3,4DAA might be prompted to differentiate into Treg cells throughupregulation of the transcription factor Foxp3 (26). In accordance withthis hypothesis we found that 3,4 DAA promoted a dose-dependent increasein the frequency of CD4+Foxp3+ T cells after stimulation of naïve CD4+ Tcells in vitro (FIG. 9E). This effect was enhanced in the presence ofincreasing concentrations of 3,4 DAA even though proliferation of the Tcells was significantly inhibited. By contrast, CA did not appear topromote Foxp3 upregulation in any of the experiments performed (FIG.9E).

CA Suppresses the Development of Autoimmunity In Vivo

IDO and the various metabolites generated by IDO have been found to havepotent immunosuppressive properties in numerous settings. In particular,3,4 DAA has been found to abrogate the development of experimentalautoimmune encephalomyelitis (EAE) in mice, and treatment of mice with3,4 DAA and 3-HKA induced similar cytokine profiles (25). Our in vitrofindings suggest that 3,4 DAA and CA may have opposing affects on immuneresponses, not unlike what has been reported for AHR ligands TCDD andFICZ. In EAE, TCDD was found to be protective by inducing Foxp3+ Tregcells, whereas FICZ increased disease severity by promoting thedifferentiation of Th17 cells, many of which also produced IL-22 (15).However, TCDD and FICZ are both known AHR agonists, like CA, whereas 3,4DAA appears to act as an antagonist. Therefore the regulation of theTh17/Th22/Treg cell differentiation pathways at the level of the AHR islikely to be more complex than merely reflecting the presence of anagonistic or antagonistic ligand. To assess whether CA was protective inEAE we performed a series of experiments in which CA was administered ina single dose (1 mg/mouse) contained within the emulsion used to inducedisease. In 4/4 experiments we found that this treatment resulted in asignificant reduction of disease occurrence and severity (FIG. 10A). Weobserved a greatly reduced number of infiltrating T cells in the centralnervous system (CNS) as well as a decrease in the frequency ofinfiltrating Th17 and Th22 cells. In contrast we observed an increase inthe frequency and number of Th17 and Th22 cells in the spleen,suggesting that CA did not inhibit T cell activation or differentiationin response to administration of MOG peptide and adjuvant. Additionallywe found that restimulation of splenocytes with MOG peptide resulted ina significantly greater amount of proliferation from CA treated micefurther demonstrating that no defect in T cell activation was noted inCA treated mice. We also observed a slight, but significant, increase inFoxp3+ Treg cells in the spleen (but not in the CNS) (FIG. 10B). In sum,we find that a single administration of CA could almost completely blockthe development of EAE in mice and that this inhibition was associatedwith a dramatic reduction in the number of T cells present in the CNS aswell as a reduction in the frequency of Th17 and Th22 cells in the CNS,but not in the periphery. These findings suggest that CA might representan excellent therapeutic agent in that it suppressed autoimmunity in theabsence of global immunosuppression.

Discussion

Here we have identified CA as an endogenous ligand of the AHR that isdirectly involved in modulating the immune response and is conservedacross species. The regulation of the generation of CA, in relation tothe metabolism of 3-HAA to PA or QA, is not completely understood. Thereare descriptions of enzymatic pathways that can result in the generationof CA from 3-HAA including the enzymes ceruloplasmin (32), superoxidedismutase (33), cinnabarinate synthase (34), and the fungal virulencefactor laccase (35). It is likely that CA could also be generatedthrough non-enzymatic reactions that would occur under oxidizingconditions (36), such as those that arise during inflammatory responses.In this regard it is interesting that reactive oxygen species (ROS)generated by neutrophils were found to be critical for IDO activationduring pathogenic fungal infections in a mouse model of chronicgranulomatous disease (CGD) (10). In this report, ROS were ascribed anessential role in the generation of superoxide, which is a co-factor forIDO and thus acts upstream of 3-HAA. However, it is likely that ROSgenerated by phagocytes could also impact the metabolism of 3-HAA,potentially leading to an increase in the generation of CA. Although ROShave typically been regarded as harmful byproducts of inflammation,there is growing evidence that they may also play a protective role inmany autoimmune diseases (37). Mutations in the gene encoding p47-phox(NCF1), which regulates oxidative burst in neutrophils, lead to thespontaneous generation of rheumatoid arthritis (RA) (38) and exacerbateEAE in mice due to elevated frequencies of autoreactive T cells (39).Neutrophils have been found to express high levels of IDO in the settingof fungal infections, and it is possible that co-expression of IDO andenzymes involved in generating ROS would strongly favor the generationof CA over PA or QA (10).

It is intriguing to hypothesize that CA may act as an alternativepathway that regulates the inflammatory response, or the damageinflicted as a result of inflammation, through activation of the AHR. Itis known that AHR−/− mice show elevated levels of inflammation inresponse to environmental pollutants suggesting that the AHR is ananti-inflammatory pathway. The generation of Th22 cells in response toAHR activation is congruent with this hypothesis. Although IL-22 wasinitially linked to IL-17 as a pro-inflammatory cytokine, recentevidence suggests that it probably plays an anti-inflammatory role onnon-hematopoietic cells, including the regulation of epithelial cellhomeostasis in the mucosal tissues. Thus the pathways that lead to thegeneration of CA may operate in tandem with the already describedimmunosuppressive mechanisms linked to tryptophan metabolism to helpgenerate a population of Th22 cells which may play a specific role intissue repair following inflammation.

Both IDO and the AHR pathway are highly conserved across evolution. Theidentification of CA as downstream metabolite of IDO capable of bindingthe AHR provides one of the first examples of an evolutionarilyconserved pathway capable of generating an endogenous AHR ligand. Thesefindings allow for future investigation into the potential roles that CAmay play in numerous biological settings in which the AHR is involved.Furthermore, we believe that therapeutic strategies targeting thegeneration of CA, or involving the direct administration of CA, mayprove useful in a variety of settings ranging from cancer to autoimmunedisease.

While specific examples have been provided, the above description isillustrative and not restrictive. Any one or more of the features of thepreviously described embodiments can be combined in any manner with oneor more features of any other embodiments in the present invention.Furthermore, many variations of the invention will become apparent tothose skilled in the art upon review of the specification. The scope ofthe invention should, therefore, be determined not with reference to theabove description, but instead should be determined with reference tothe appended claims along with their full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument, Applicants do not admit any particular reference is “priorart” to their invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference. Where a conflictexists between the instant application and a reference provided herein,the instant application shall dominate.

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What is claimed is:
 1. A method of treating an immune disorder, themethod comprising administering to a subject in need thereof, atherapeutically effective amount of a compound of formula Ib:

wherein each R¹ is independently selected from the group consisting ofhydrogen, —NH₂, C₁₋₆ alkylamine, —CO₂H, C₁₋₆ alkyl-CO₂H, and C₀₋₆alkyl-C(O)NH₂; each R² is independently selected from the groupconsisting of hydrogen, —NH₂, C₁₋₆ alkylamine, —CO₂H, C₁₋₆ alkyl-CO₂H,and C₀₋₆ alkyl-C(O)NH₂; subscript m is an integer from 1 to 3; subscriptn is an integer from 1 to 4; and salts and isomers thereof, therebytreating an immune disorder.
 2. The method of claim 1, wherein thecompound of formula Ib is:


3. The method of claim 1, wherein the immune disorder is an autoimmunedisorder selected from the group consisting of multiple sclerosis,myasthenia gravis, Guillan-Barre syndrome (antiphospholipid syndrome),systemic lupus erytromatosis, Behcet's syndrome, Sjogrens syndrome,rheumatoid arthritis, Hashimoto's disease/hypothyroiditis, primarybiliary cirrhosis, mixed connective tissue disease, chronic activehepatitis, Graves' disease/hyperthyroiditis, scleroderma, chronicidiopathic thrombocytopenic purpura, diabetic neuropathy and septicshock.
 4. A method of modulating Aryl Hydrocarbon Receptor activity byadministering a compound of formula Ib:

wherein each R¹ is independently selected from the group consisting ofhydrogen, —NH₂, C₁₋₆ alkylamine, —CO₂H, C₁₋₆ alkyl-CO₂H, and C₀₋₆alkyl-C(O)NH₂; each R² is independently selected from the groupconsisting of hydrogen, —NH₂, C₁₋₆ alkylamine, —CO₂H, C₁₋₆ alkyl-CO₂H,and C₀₋₆ alkyl-C(O)NH₂; subscript m is an integer from 1 to 3; subscriptn is an integer from 1 to 4; and salts and isomers thereof, therebymodulating Aryl Hydrocarbon Receptor activity.
 5. The method of claim 4,wherein the compound of formula Ib is: